(20S)-(+)-Camptothecin, a pyrrolo [3,4-b] quinoline alkaloid, is the active principle of the Chinese tree, Camptotheca acuminata Decne, extracts of which have been used for centuries in traditional Chinese medicine to treat cancer. The alkaloid is mostly found in the bark of the plant, a fast growing deciduous tree native only to China and Tibet, where it is known as xi shu ("happy tree").
Camptotheca belongs to the family Nyssaceae (tupelo family) and may reach a height of about 25 m. It was first introduced to the United States in 1911 as an ornamental plant and on several occasions subsequently. More recently, C. acuminata plantations were established in southern Louisiana in an effort to provide raw plant materials for the production of camptothecin. The alkaloid may also be obtained from trees of the species Nothapodytes, which are native to the Indian subcontinent.
1.1 Scientific Interest in CPT
CPT was first isolated by Wall et al. in 1966 during an antitumor screening program, and immediately it generated a great deal of interest, both in the clinical and the chemical arenas, as a new agent for the treatment of human malignancy. The compound itself is highly insoluble in aqueous media, but the sodium salt, obtained by alkaline hydrolysis of the lactone ring, displayed good water solubiliy and was easily formulated for intravenous administration. As a consequence, phase I clinical trials were launched in the early 1970's in patients with an advanced form of gastrointestinal cancer. In retrospect, those protocols were poorly designed (vide infra), but in any event they led to the erroneous conclusion that CPT was only marginally effective. The drug did produce partial remissions in some patients, but the trials were eventually halted because of severe toxic side effects.
1.2 Mechanism of Action: Inhibition of Topoisomerase I
Details of the mechanism of action of CPT remained a mystery until 1985, when Liu et al. discovered that the substance is a specific inhibitor of topoisomerase I (Topo I), an essential enzyme for DNA replication and transcription. Topo I is a monomeric protein with a MW of ca.100 kD that relaxes (i.e., unwinds) torsionally strained (supercoiled) DNA ahead of active transcription and translation sites. The enzyme initially binds noncovalently to double-stranded DNA, but then it creates a transient break in one strand and concomitantly becomes covalently bound to the 3'-phosphoryl end of the nicked nucleic acid strand. The unbroken DNA strand is allowed to unwind once and to pass through the break site, before topo I religates the cleaved DNA and reestablishes the double-stranded configuration These events constitute an obligatory stage of DNA replication and transcription, as the chromosomes must be unwound in order for the cell to express genetic information or to divide. The covalent complex between topo I and a single DNA strand, also termed the "cleavable complex", is in rapid kinetic equilibrium with the noncovalently bound complex (the "noncleaveable complex"). Camptothecin is believed to express its activity by reversibly binding to the cleavable complex and stabilizing it, thereby inhibiting religation of the nicked DNA strand. As a consequence, the advancing DNA polymerase operating in the replicating fork soon "collides" with the stabilized cleavable complex and creates an irreparable double-strand break, which is fatal to the cell.
Cancer cells are more vulnerable to topoisomerase inhibition than normal cells, because they grow and reproduce at a much faster rate. Even more significantly, elevated concentrations of topo I have been found in many different tumor cells. In 1994, CPT was also found to inhibit both acute and chronic HIV-I infections. These observations have stimulated renewed interest in camptothecin, and as a result, much research in its pharmacology, medicinal chemistry and total synthesis is currently underway. Camptothecin itself has been used in China to treat leukemias and carcinomas of the stomach and liver, but it is not is not approved by FDA for use in the US. Considerably more promising are a number of semisynthetic derivatives currently being tested in phase I and phase II clinical trials. Examples include Gl 147211C, irinotecan (CPT-11), 9-aminocamptothecin (9-AC), 9-amino-10,11-methylenedioxy camptothecin (9-AC-10,11-MD) and topotecan. The latter substance was approved by FDA in May 1996. Positive results have recently been reported in the treatment of diverse neoplasms, such as colon and breast cancers, malignant melanoma, small-cell lung cancer, ovary, leukemia and non-Hodgkin's lymphoma. Toxicity remnains a problem, major side effects including severe diarrhea, nausea, leukopenia, and possibly bone marrow depression.
1.3 Relationship Between CPT Structure and Activity
The relationship between the structure of CPT and in vitro and in vivo activity has been reported in detail, hence this section will summarize only major findings.
1. An intact E ring (lactone moiety) is necessary for activity.
2. The (20S) configuration is an absolute requirement, (20R)-CPT or analogs being inactive in vitro and in vivo.
3. With the exception of SN-38, only ring A derivatives increase antitumor activity. Substitutions at C-9 or C-10 generally enhance activity.
4. The pentacyclic structure of CPT is an absolute requirement. Tetracyclic analogs or bicyclic and tricyclic analogs are inactive.
5. Disubstitution in general leads to CPT analogs with reduced or no activity, the exception being the 10,11-methylenedioxy moiety, which greatly increases activity. Substitution in the 11- and 12-positions gave CPT analogs which exhibited reduced activity or total inactivation.
6. The a-hydroxy lactone moiety in ring E is required.
7. Substitution of nitrogen for OH or lactone oxygen in ring E leads to inactivation.
8. The pyridone ring D is required. Replacement of the pyridone ring by an aromatic ring results in inactivation.
9. The C-20 ethyl substituent is required, although there is some flexibility; for example, substitution of a 20-allyl group resulted in good activity, whereas substitution of a methyl group led to inactivation.
1.4 Biosynthetic Pathway
In 1967 Wenkert et al. hypothesized that CPT is derived biosynthetically from tryptophan and secologanine. Winterfeld later expanded on this idea based on his own finding that indole derivatives underwent facile auto-oxidation to quinolones in vitro.
The proposed biosynthetic pathway of CPT was later verified and further elaborated by Hutchinson via plant feeding experiments with regiospecifically labeled precursors. It seems clear that strictosamide is a key precursor. Transformation of strictosamide into CPT is considered to be possible through ring BC oxidation followed by recyclization, ring D oxidation, removal of the C-21 glucose moiety and ring E oxidation.
1.5 Previous Syntheses
Further clinical evaluation of CPT, and congeners is intimately dependent on the availability of an efficient total synthesis. The natural product is exceedingly rare and costly. It may be obtained virtually exclusively from the People's Republic of China through unreliable suppliers authorized by the Chinese Government. Large orders (100 g) are priced at ca. $ 35-40/g. The latest (1996-97) catalog from the Aldrich Chemical Co. lists CPT for $ 127.50/g: nine times the current price of pure gold ($ 395/oz=$ 14.10/g). In recent times it has become apparent that certain derivatives of CPT possess more desirable pharmacological properties. The preparation of these substances by semisynthesis is often lengthy and inefficient, because the natural product does not lend itself readily to derivatization, and because its insolubility in most organic solvents creates a host of technical problems. In this light, total synthesis appears to be an entirely acceptable alternative, if not a much better approach. Yet the deceptively simple structure of camptothecin is endowed with robust defenses against a synthetic attack, not the least of which is the crucial configuration of the C-20 center. Despite the fact that twenty-five years have elapsed since the first total synthesis of CPT was accomplished, practical avenues have emerged only beginning in 1994. It would be inappropriate to discuss all the numerous published syntheses of CPT in detail, since comprehensive reviews are available. We shall survey instead only the most significant efforts, with the focus on recent enantioselective routes.
The first total synthesis of (.+-.)-CPT was reported by Stork and Schultz in 1971. A key transformation was a base-catalyzed Friedlander condensation of pyrrolidinone with 2-aminobenzaldehyde to give the tricyclic pyrrolo [3,4-b]quinoline acid. This was converted to the tetracyclic b-ketolactam, which was advanced to (.+-.)-CPT. Racemic CPT was thus prepared in 17 steps and in ca. 1-2% overall yield.
Danishefsky recently improved his 1971 synthesis of (.+-.)-CPT, the second one ever to be published, to 9 steps and 31% yield starting from vinylogous urethane, through intermediacy of the key tricyclic lactone. A key strategic idea is the formation of the lactone by hydroxymethylation of the pyridone derived from, a transformation that is poorly regioselective unless one of the reactive sites of that heterocycle is blocked. This function is delegated to a carbomethoxy group occupying a position corresponding to C-14 of CPT. That functionality also increases the acidity of protons on the vinylogous CH2 group (C-2 in CPT), thereby facilitating the subsequent introduction of a ketone required for Friedlander reaction.
Winterfeldt discovered in 1971 that indoles are autoxidized to quinolones in strongly basic dimethylformamide solution. The ease with which this transformation occurred led to the hypothesis that quinolones may be biosynthetic intermediates. Several of the proposed precursors were subsequently isolated from plant extracts.
An imaginative total synthesis of CPT was readily developed on the basis of this facile interconversion, which in the specific case evolved from triester. Racemic CPT was thus obtained in 13 steps and 12% yield from the known ester.
The first synthesis of (+)-camptothecin was announced in 1975 by Corey. A characteristically elegant synthetic design resting on the merger of chiral pseudoacid chloride with tricyclic amine was ultimately marred by poor yields (0.3% overall) and lengthy sequences (21 steps). Scalemic lactone was secured through resolution of the racemate via its diastereomeric quinine salts, followed by treatment with methyl chloroformate, and removal of the quinine salt by quaternization with methyl iodide.
Resolution was also a central feature in the Wall synthesis of (+)-CPT. Pyridone was prepared by condensation of 2-cyanoacetamide with the enol ether of ethyl acetpyruvate and advanced to in 12 steps and 14% yield. Resolution was conducted by cleavage of the lactone ring with (S)-(-)-a-methylbenzylamine, separation of the diastereomers, and relactonization. The loss of at least one-half was still a drawback.
The first attempt to produce a scalemic CPT intermediate by means other than resolution was reported by Tagawa. The cornerstone of this approach is a diastereoselective ethylation of the enolate. A chiral auxiliary derived from D-proline (expensive--$ 16/g from the Aldrich Chemical Co.) induced an 82:18 diastereoselectivity in favor of the desired stereoisomer, which was recrystallized to optical purity. Camptothecin was therefore reached in eight steps and 19% yield, which itself resulted from a multistep sequence as reported earlier by Wall. The overall yield was therefore around 7% over no less than 14 steps.
The best enantioselective synthesis of camptothecin known prior to our work was reported in 1994 by Fang and collaborators at the Glaxo Pharmaceutical Company. Their approach is conceptually similar to a route described by Comins, with whom the Glaxo group has had a long standing collaboration on the CPT problem, but it differs from the Comins plan in that asymmetry is created by a Sharpless dihydroxylation instead of a diastereoselective carbonyl addition. A common theme remains the Comins metallation technology for the elaboration of the central pyridone unit.
The Comins synthesis (3% overall yield for 8 steps) elaborated a "stripped down" pyridone to iodide, which was lithiated (n-BuLi) and treated with the 2-ketobutyrate ester of (-)-trans-2-(a-cumyl)-cyclohexanol to afford in 88% diastereomeric excess. A drawback of this approach is that the chiral auxiliary does not appear to be commercially available; nevertheless, it is recoverable and recyclable. The ring ABC triad was installed in an elegant sequence involving N-alkylation followed by radical cyclization.
The Fang strategy centers on asymmetric Sharpless dihydroxylation. The creation of this pyridine relies largely on Comins technology, with an intramolecular Heck reaction as an additional interesting twist. Rings ABC were introduced also as shown earlier by Comins, except that an intramolecular Heck reaction was preferred to a radical cyclization.
An even more recent enantioselective synthesis of CPT has been disclosed by Curran. No conceptual advances are evident in this work with respect to the issue of enantioselectivity; indeed, the key intermediate was prepared in a manner identical to Fang's. However, an elegant cascade of radical processes was utilized to complete rings B and C of the natural product. Highly enantiomerically enriched CPT (95% ee) thus emerged in 9 steps and 3% overall yield from pyridine.